Preventing sexual transmission of human immunodeficiency virus (HIV-1) is critical for altering the course of the global pandemic of acquired immunodeficiency syndrome (AIDS). Currently, approximately 34 million people are living with HIV-1 infection; 2.5 million people are newly infected with the virus annually, and nearly 1.7 million individuals succumb each year to AIDS. Hence, there is an urgent need to develop strategies that can prevent HIV-1 transmission.
Targeting the early phase of HIV-1 infection, including virus entry, as a prophylactic modality is a focus of intense research. HIV-1 entry involves a series of events that include attachment to the host cell and fusion of the viral and target cell membranes. HIV-1 entry is mediated by the viral spike, which is composed of three gp120 envelope glycoproteins and three gp41 transmembrane molecules. In humans, HIV-1 infection begins with two consecutive gp120 binding events, each associated with major conformational changes in the gp120 structure. The first involves gp120 binding to the host CD4 receptor. CD4 binding results in a major gp120 conformational change, thus exposing a site for binding to the chemokine receptor (either CCR5 or CXCR4). Chemokine receptor binding is accompanied by gp41 rearrangement and the insertion of the gp41 fusion peptide into the host cell membrane, permitting fusion and viral entry. The highly conserved gp120-CD4 interface has been revealed by a number of X-ray crystal structures of the gp120 core domain, complexed to the D1D2 fragment of CD4 and a Fab of a human neutralizing antibody 17b, the latter serving as a surrogate for the co-receptors. CD4 binding induces the formation of a large internal cavity at the interface of the three gp120 domains, the inner domain, the outer domain, and the bridging sheet domain. The Phe43CD4 and Arg59CD4 residues have been shown by both mutagenesis and structural studies to be critical for binding of gp120 to CD4. Residue Phe43CD4 is located on the CD4 CDR2-like loop and binds at the vestibule of the large cavity formed upon the CD4-induced gp120 conformational change; Arg59CD4 is located on a neighboring β-strand and forms an electrostatic interaction with Asp368gp120 at the cavity vestibule. The structure of the unbound form of the simian immunodeficiency virus (SIV) gp120, which has a 35% sequence identity with HIV-1 gp120, indicates an invariant outer domain, with conformational changes occurring in both the bridging sheet and inner domain. Recent studies indicate that the HIV-1 gp120 core exhibits a propensity to assume the CD4-bound conformation, but is restrained from doing so by gp120 variable loops and interactions with gp41 in the context of the trimer spike. The thermodynamic signature of the CD4-induced gp120 conformational change exhibits a highly favorable binding enthalpy balanced with a highly unfavorable entropy associated with molecular ordering.
Two N-phenyl-N′-(2,2,6,6,-tetramethyl-piperidin-4-yl)-oxalamide compounds, NBD-556 and NBD-557, were identified via screening a drug-like small-molecule library for inhibition of gp120-CD4 binding. Mutagenesis, modeling and synthesis of NBD analogues with improved binding affinity revealed that these small molecules bind to the highly conserved gp120 cavity and compete with CD4 binding. Exploration of structure-activity relationships (SAR) demonstrated that compounds with comparable binding affinities act both as CD4 antagonists (i.e., to inhibit HIV-1-infection of CD4+ cells) and as CD4 agonists (i.e., promote CCR5 binding and enhance viral infection in the absence of CD4). Mimicry of CD4 was further demonstrated by the similarity of the NBD and CD4 thermodynamic signatures, both exhibiting a large unfavorable entropy change, −TΔS, to Gibbs energy (17.1 kcal/mol and 24.1 kcal/mol for NBD-556 and CD4, respectively) compensated by a large favorable enthalpy change (−24.5 kcal/mol and −34.5 kcal/mol for NBD-556 and CD4, respectively). Taken together, these results provided a rationale for further optimization of NBD analogues as inhibitors of HIV-1 viral entry by focusing on both Phe43 cavity and Asp368gp120 hotspots.
There exists a need for small molecule inhibitors exhibiting improved thermodynamic and antiviral properties that are useful in treating or preventing HIV.
In addition to small molecule inhibitors of HIV, HIV-1-neutralizing antibodies are an important component of a protective vaccine-induced immune response. Passive administration of HIV-1-neutralizing antibodies protects monkeys from intravenous and mucosal challenge with simian-human immunodeficiency viruses (SHIVs). The trimeric envelope glycoprotein (Env) spike on the virion surface is the only HIV-1-specific target accessible to neutralizing antibodies. The presence of circulating antibodies against a specific region of Env (the gp120 V2 variable region) correlated with the partial protection seen in the RV144 clinical vaccine trial. Thus, the generation of anti-Env antibodies, particularly neutralizing antibodies, may be critical for a successful HIV-1 vaccine.
The HIV-1 Env spike described above, which is composed of three gp120 exterior Envs and three gp41 transmembrane Envs, mediates virus entry into host cells. The unliganded HIV-1 Env is metastable. Binding of gp120 to the initial receptor, CD4, triggers Env conformational changes that result in the formation/exposure of two elements: 1) the gp120 binding site for the second receptor, CCR5 or CXCR4, and 2) the gp41 heptad repeat (HR1) coiled coil. Binding of gp120 to the CCR5 or CXCR4 coreceptor is thought to induce further Env conformational changes that result in the formation of an energetically stable gp41 six-helix bundle that promotes the fusion of the viral and target cell membranes.
As a successful persistent virus, HIV-1 has evolved Env spikes that minimize the elicitation and impact of neutralizing antibodies. These features include surface variability, conformational lability and a heavy coat of glycans. Most anti-Env antibodies elicited during natural infection do not neutralize HIV-1, and those that do are usually strain-restricted, allowing virus escape. Only after several years of infection in some HIV-1-infected individuals are more broadly neutralizing antibodies generated. Broadly HIV-1-neutralizing antibodies typically display unusual features that allow binding to the heavily shielded, conserved Env epitopes. Some neutralizing antibodies with modest breadth bind Env carbohydrate-dependent epitopes. The variable and glycosylated features of the HIV-1 Env spike render the elicitation of neutralizing antibodies difficult, and have presented extreme challenges to the development of effective Env vaccine immunogens. Even the best current HIV-1 Env immunogens elicit antibodies that inhibit the infection only of the small subset of primary viruses that are more prone to neutralization. The sensitivity of HIV-1 strains to antibody neutralization depends upon the integrity of the Env epitope and Env reactivity; the latter property indicates the propensity of unliganded Env to undergo conformational changes. A successful HIV-1 vaccine must cover a range of phylogenetically diverse transmitted/founder viruses, most of which have Envs of low reactivity and thus exhibit low sensitivity to neutralization by antibodies.
One of the major hurdles facing the development of a successful HIV-1/AIDS vaccine is the requirement to elicit antibodies that recognize conserved elements of the native, unliganded conformation of the HIV-1 Env trimer. These conserved elements are often buried or composed partially or in some cases completely of glycans, which render the generation of the cognate antibodies inefficient. Two functionally conserved gp120 elements interact with the HIV-1 host cell receptors, CD4 and CCR5/CXCR4. The CD4-binding site (CD4BS) on gp120 is sterically recessed on the HIV-1 Env trimer and surrounded by regions that exhibit inter-strain variability and glycosylation. Effective neutralizing antibodies directed against the gp120 CD4BS typically engage their epitopes in a manner that does not require the Env trimer to undergo significant conformational changes. Indeed, potently neutralizing antibodies directed against multiple conserved HIV-1 Env epitopes generally require minimal conformational change in the unliganded Env trimer for their binding.
The vast majority of primary HIV-1 isolates, including transmitted/founder viruses, use CCR5 as a second receptor. The CCR5-binding site on gp120 consists of a discontinuous surface of the gp120 core and the tip of the V3 loop, both of which are well conserved among primate immunodeficiency viruses. These elements are not formed and exposed on HIV-1 Env trimers with low envelope reactivity. Antibodies that recognize CD4-induced (CD4i) epitopes in the gp120 core bind near or within the coreceptor-binding site of gp120. Some of these antibodies are specific for CCR5-using HIV-1 variants, whereas other antibodies recognize both CCR5-using and CXCR4-using viruses. CD4i antibodies are routinely generated in HIV-1-infected humans, and can be elicited by HIV-1 gp120 core constructs in which the CD4-bound conformation has been stabilized by disulfide bonds and cavity-filling substitutions. Although both the CD4i epitopes and the V3 tip become exposed after HIV-1 binding to cell-surface CD4, steric factors (e.g., the target cell membrane) limit the ability of CD4i and V3-directed antibodies to bind their respective epitopes and neutralize the virus. Therefore, the neutralizing potency of CD4i and V3-directed antibodies is related to the degree of exposure of these epitopes on the unliganded Env trimer. Thus, because of the low Env reactivity of primary and transmitted/founder HIV-1, these viruses are generally inhibited poorly by most CD4i and V3-directed antibodies.
There exists a need for methods of eliciting antibodies that bind the unliganded HIV-1 Env trimer efficiently and neutralize the large fraction of primary transmitted/founder HIV-1 with low Env reactivity.
Furthermore, induction of the CD4-bound conformation renders primary HIV-1 sensitive to neutralization by CD4i antibodies. HIV-1 sensitization as a strategy for virus prophylaxis has become feasible as a result of the availability of small-molecule CD4-mimetic compounds. As mentioned above, NBD-556 and NBD-557, were discovered in a screen for inhibitors of gp120-CD4 interaction. NBD-556 and NBD-557 bind in the Phe 43 cavity, a highly conserved ˜150 cubic Angstrom pocket in the gp120 glycoprotein of all HIV-1 strains except those in Group O. The vestibule of the Phe 43 cavity contains a number of conserved gp120 residues that make critical contacts with CD4. The binding of NBD compounds in the Phe 43 cavity blocks gp120-CD4 interaction and, like the binding of soluble CD4, prematurely triggers the activation of the HIV-1 Env spike. The activated state is short-lived (t1/2=5-7 minutes at 37° C.) and the bound Env spike rapidly decays into an irreversibly inactivated state. Although NBD-556 induces large, entropically unfavorable changes in gp120 conformation and thus binds with only modest affinity (Kd=3 μM), iterative cycles of co-crystallization with gp120 and rational design and synthesis have yielded a number of NBD-556 analogues with improved affinity and antiviral properties. However, NBD-556 suffers from one significant disadvantage with respect to development of a vaccine: it increases the binding or neutralizing potency of the 17b CD4i antibody weakly and only in laboratory-adapted viruses that have high Env reactivity.
There also exists a need for a method of increasing the sensitivity of the HIV-1 virion to antibody neutralization.